DNA sequence-based HLA class I typing method

ABSTRACT

The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of cDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of HLA-A, HLA-B, and HLA-C transcripts enzymatically amplified and sequenced using a limited number of selected oligonucleotides.

This invention was made with Government support under grant number DK36828 by the U.S. National Institutes of Health. The Government hascertain rights in the invention.

TECHNICAL FIELD

The present invention relates to a process for determining genotypes ofhighly polymorphic systems, such as the Class I genes of the majorhistocompatibility complex of humans. Specifically, the method of thepresent invention involves amplifying the alleles carried by any givenindividual at a gene locus or loci of interest by polymerase chainreaction with selected oligonucleotide primers. The polymerase chainreaction products are directly sequenced followed by evaluation of theresulting nucleic acid ladders to determine the genotype of samplenucleic acid.

BACKGROUND OF THE INVENTION

HLA Class I Polymorphism Analysis.

1. Introduction

The major histocompatibility complex (MHC) includes the human leukocyteantigens (HLA) gene complex which is located on the short arm of humanchromosome six. These genes encode cell-surface proteins which regulatecell-cell interactions of the immune response. The various HLA Class Iloci encode the heavy chain of the Class I molecules (44,000 daltonpolypeptide), which associate with B-2 microglobulin. The differentClass I dimers are expressed on the surface of essentially all humancell types. These molecules are involved in the presentation ofendogenous peptides (i.e., virally-encoded proteins) by the expressingcells; this molecular complex is recognized on the target cells bycytotoxic T lymphocytes, in a self-restricted manner.

The HLA-A, -B and -C loci of the HLA Class I region exhibit anextraordinarily high degree of polymorphism. The WHO nomenclaturecommittee for factors of the HLA system [Marsh and Bodmer,Immunogenetics, 31:131 (1990)]designated 25 alleles at the HLA-A(HLA-A,0101, A*0201, etc.), 32 alleles at the HLA-B, and 11 alleles atthe HLA-C loci. Since this high degree of polymorphism relates to thefunction of the HLA molecules, much effort has gone into designingaccurate and optimal methods for detecting the many possible allelesthat may be carried at each locus by any given individual of thepopulation. Products of the HLA genes were first identified by reactionsof antisera. Serological techniques remain the primary, and in manycases the only, typing method for HLA antigens. The complement-dependentcytotoxicity (CDC) assay is the method most often used to defineserologic specificities [Terasaki and McClelland, Nature, 204:998,(1964)]. The advantages of the CDC include the small volumes of antiseraand target cells and the relatively short time required for the test.

One great disadvantage of serological typing is that alloantisera arenot infrequently limited in number and volume, and often have complexreactivities, so that an extensive program is required to identify andobtain useful sera. Perhaps most importantly, however, many cellularlyand/or biochemically defined polymorphisms, which in all likelihood havefunctional significance in bone marrow transplantation, cannot bedetected by serological techniques [Anasetti et al., Hum. Immunol.,29:70 (1990)].

One-dimensional isoelectric focusing (1D-IEF) is very effective inidentifying serologically undetectable variants or subtypes for theHLA-A and HLA-B antigens [Yang, Immunobiology of HLA, Vol. I, 332(1989)]which have been implicated in the development of acutegraft-versus-host disease and in graft rejection in bone marrowtransplantation. In this technique, metabolically labeled cell lysatesare immunoprecipitated using monoclonal antibodies to Class I antigensand the immunoprecipitates are desialated and subjected toisoelectricfocusing. The technique, however, is time consuming andcumbersome. Interpretation of the IEF patterns is dependent on priorknowledge of the serological definition, and certain HLA antigens showso many overlapping bands that IEF assignment is difficult andimpractical.

It is believed that only about 30% of the existing Class I sequences areknown as of today. This limited knowledge extraordinarily complicatesthe application and use of other molecular approaches for Class I HLAtyping which are being used for Class II typing (i.e., oligotyping).Furthermore, because the distribution and nature of the sequencepolymorphisms in the Class I genes is very different from that seen inClass II genes, oligotyping strategies for Class I genes may require theuse of a very large number of oligonucleotides to type the alleles knownat the present time and this number may become much larger as newallelic sequences are described. Application of a sequence-based typingtechnique to the analysis of HLA Class I polymorphism presented a seriesof problems additional to those associated with HLA Class II typing.These problems are: 1) the informative polymorphism at Class I locispans 2 different exons (approximately 600 base-pairs), instead of 1 inClass II genes; 2) the HLA-A, -B and -C loci are believed to be far morepolymorphic than Class II loci; 3) the different Class I loci are farmore related to each other in terms of DNA and amino acid sequence thanClass II loci are to each other. Thus, when the alleles at each locusare compared with each other, they resemble alleles at a single locusand, therefore, given that up to 6 different Class I genes may beexpressed by a given individual (2 alleles at each locus) this increasesthe number of technical limitations to molecular typing imposed by thispolymorphic system.

Accordingly, there is a need for a method to determine genomicinformation from such a highly polymorphic system as the HLA- Class Isystem that addresses the limitations imposed by previous methods. Thatis, a system that is capable of determining the nucleotide sequences ofthe genes carried by any given individual without the need to haveprevious knowledge of the individual's HLA type as defined by othermethods.

The present invention provides a molecular approach for accurate HLAClass I sequence-based typing that is rapid, avoids the use ofoligonucleotides specific for each known allele, requires the use ofonly a small number of oligonucleotide primers, does not requireprevious typing information, can readily detect new sequence variantsunidentifiable with more conventional approaches, and is entirelyautomatable.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining the nucleicacid sequence of one or more polymorphic genes of a subject byamplifying and direct sequencing genomic or complementary DNA moleculesfor each allele at each gene locus to be sequenced. The amplificationand sequencing of DNA molecules utilizes selected locus-specificoligonucleotide primers that provide for detailed characterization ofHLA gene polymorphism, at the sequence level, in the population. Themethod is specifically designed to provide rapid and accuratedetermination of a major histocompatibility complex class genotype of asubject in a sample (e.g., Class I). Most particularly, the method isdirected to determining at least one HLA Class I gene locus includingHLA-A, and/or HLA-B, and/or HLA-C genes.

To determine a Class I gene locus nucleic acid sequence polymorphismwith the method of the present invention, nucleic acid (RNA or DNA) froma sample is isolated. According to the present invention, the samplenucleic acid sequence is determined by: amplifying the cDNA molecules orgenomic DNA by polymerase chain reaction to generate sufficient productfor each allele of each gene locus to be sequenced, with all of thealleles for each gene locus and chromosome to be sequenced beingamplified with at least one Class I loci-specific primer annealing toall possible alleles at all Class I loci at each chromosome and alocus-specific primer that anneals preferentially to a region of eachsaid gene locus which has a sequence that is shared by all alleles atsaid locus; preparing the products of each PCR for sequencing (clean);sequencing directly the products of each polymerase chain reactionproduct to detect each allele at each gene locus of each chromosome withTaq polymerase and either a locus-specific primer and a Class Iloci-specific primer or two Class I loci-specific oligonucleotideprimers; and analyzing each sequenced product for each locus and primercombination(s) to determine the genotype of the subject. The analysis isconducted by comparing the nucleotide sequence of each allele of eachgene locus sequence to known sequences for each locus. Comparison ofnucleic acid ladders for sequenced alleles can be conducted visually orusing computer software.

In a preferred embodiment of the present invention the nucleic acid isRNA. When the subject nucleic acid is RNA, prior to amplification, cDNAmolecules are synthesized for each allele at each Class I loci of eachchromosome to be sequenced using oligonucleotide primers that anneal toa region of each gene locus shared by all alleles of each said genelocus. The unincorporated oligonucleotide primer used for generating thecDNA is then removed.

In a preferred embodiment, the process of the invention is automated foruse in rapid genotype determinations, including diagnosis of geneticdiseases, forensics and paternity testing. Automation of the processincludes isolating the sample nucleic acid with an RNA/DNA extractor;amplifying the synthesized cDNA molecule or the isolated DNA molecule bypolymerase chain reaction using a thermocycler to generate thepolymerase chain reaction products; sequencing the polymerase chainreaction products in an automated sequencing apparatus; and analyzingeach sequenced polymerase chain reaction product with the computerhaving a database with allelic sequence information and the capacity toconduct the appropriate algorithm for comparing the polymerase chainreaction product sequence for each allele amplified with known genesequences.

The invention further relates to specific groups of oligonucleotideprimers useful in the steps of cDNA synthesis, cDNA amplification bypolymerase chain reaction and direct sequencing of the polymerase chainreaction products to determine the nucleotide sequence of each of thealleles at each locus of each chromosome that is amplified. Usefulsingle strand DNA oligonucleotide primers are described in Table 1herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of the primer binding sites on HLA-A, HLA-B,and HLA-C transcripts involved in the cDNA/PCR/Sequencing steps fordetermining HLA Class I (HLA-A, HLA-B and HLA-C) genes. In FIGS. 1A-1D,stippled boxes represent RT primers; black boxes represent PCR primers;blank boxes represent sequencing primers; boxes with horizontal linesrepresent RT-PCR primers; and boxes with diagonal bars are PCR/seqprimers.

FIGS. 1B-1D show primer binding sites on HLA-A, HLA-B and HLA-Ctranscripts in the method according to the present invention. Eachfigure shows the end-products of the two reactions for each locus.

FIG. 2 shows a flow-chart of the procedure for peripheral blood samples.Each reaction is performed in a different test tube. The reactions arenamed with numbers; these numbers (1-6) correspond to those shown inTable II (combinations of primers/reaction).

FIG. 3 shows family pedigree of subjects PC, AR and KR as well as theirserological typing results for HLA-A, -B and DR loci. Sequence-basedClass I typing was not only able to predict the serological reactivitiesof these subjects, but also to identify new allelic sequences notdetectable by serology (i.e., second haplotype of KR).

FIGS. 4A and 4B show direct sequencing of A (left) and B (right) genesof subjects PC and AR from family of FIG. 3 using the combination ofprimers of reactions #2 and #4, respectively. This combination isindicated at the bottom of the Figure. For practical purposes, thetemplate codons to which each of the used primers anneal (i.e., primerABC101=ABC(-8/-1) are indicated in parenthesis. For the A locus, theFigure shows results obtained by using different MgCl₂ concentrations.By decreasing the final MgCl₂ concentration (i.e., 3 μl of 25 mM MgCl₂),the reaction results in the selective amplification of one of the twoalleles present at the A locus. Increasing the final MgCl₂ concentrationallows to see the ladders corresponding to each allele without losingthe locus-specificity of the reaction. On the side of the Figure, thepositions where two bands can be seen or where there should be two bandsaccording to the expected sequences for the serological specificitiesexpressed by these subjects is indicated. The observed (obs.) andexpected (pred.) sequences corresponding to each allele at each locusare shown. The locus-specificity of the reactions was assessed by thepresence of locus-specific bases at the positions indicated. Codonpositions are also indicated as reference points. Comp. means"Compression" and indicates the presence of a sequence artifact at thisposition due to a compression of bands in the ladder during theelectrophoresis. For the B locus, results using two differentconcentrations of PCR primers (100 ng each or 200 ng each) are shown.

FIG. 5 shows ladders generated for the A locus in 3 homozygous celllines and a heterozygous subject using combination of primers ofreaction #1 (bottom of Figure). Observed and expected sequences areindicated on the side of the Figure for the heterozygote, wherever twobands are either present or expected. Locus- and allele-specificpositions, as well as a compression artifact, are also indicated.

FIG. 6 shows ladders generated for the B locus in two homozygous celllines and a B locus homozygous subject using conditions of reaction #3.Predicted and expected sequence for specificity B38 (JBUSH cell line)are indicated.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "gene" refers to a segment of DNA, composed ofa transcribed region and a regulatory sequence that makes possible atranscription. The term "gene locus" refers to the specific place on thechromosome where a gene is located. The term "allele" refers to themultiple forms of a gene that can exist at a single gene locus at asingle chromosome and are distinguishable from the other possiblealleles by their differing effects on phenotype (detectable outwardmanifestations of a specific genotype). "Haplotype" refers to thespecific allele composition of the genes at multiple loci on the samechromosome. As used herein the term "genotype" refers to the specificallelic composition of a gene at multiple linked loci at each chromosome(2 haplotypes).

The term "oligonucleotide" as used herein refers to a molecule havingtwo or more deoxyribonucleotides or ribonucleotides, preferably morethan three deoxyribonucleotides. The exact number of nucleotides in themolecule will depend on the function of the specific oligonucleotidemolecule. As used herein the term "primer" refers to a single strandedDNA oligonucleotide sequence, preferably produced synthetically which iscapable of acting as a point of initiation for synthesis of a primerextension product which is complementary to a nucleic acid strand to becopied or a point of initiation for sequencing a DNA molecule. In thecase of primers intended for use in synthesizing cDNA or amplifying cDNAor genomic DNA molecules by polymerase chain reaction products, thelength and sequence of the primer must be sufficient to prime thesynthesis of extension products in the presence of a polymerizationenzyme. Preferably, the length of the primer is from about 5-50nucleotides, more preferably from about 5-20 nucleotides. Specificlength and sequence of the primer will depend on complexity of requiredDNA or RNA target templates, as well as conditions of primer employmentsuch as temperature, ionic strength, and MgCl₂ concentration as well asrole of primer in the reaction (cDNA synthesis, PCR or sequencing).

As used herein, "locus-specific oligonucleotide primer" refers to anoligonucleotide molecule that corresponds to a region of high DNAsequence conservation (i.e. less than 1-2 nucleotide variations) amongall alleles of a single gene locus. For example, in the case of theHLA-A locus, the locus-specific oligonucleotide primer will anneal orbind preferentially to all HLA-A alleles under the conditions describedhere. Similarly, HLA-B and HLA-C locus-specific oligonucleotide primerswill preferentially bind to HLA-B and HLA-C alleles, respectively. Incontrast to this, "Class I loci-specific oligonucleotide primer" refersto an oligonucleotide molecule that corresponds to a region of high DNAsequence conservation (i.e., less than about 1-2 nucleotide variations)among all the individual gene loci of a gene class and anneals to aregion of a group of related HLA loci that has the same conservedsequence for all alleles at all the related loci. In the preferredembodiment, the Class I loci-specific oligonucleotide primers anneal orbind to a relatively conserved (i.e., about 1-2 mismatches) region ofall alleles of HLA-A, HLA-B, and HLA-C genes. While the Class I loci- orindividual locus-specific primer need not correspond exactly to thenucleotide template to which it anneals, as stated above, each primerwill have minimal, preferably less than one or two mismatches with thetarget nucleotide template. Functionally, the Class I loci- andindividual locus-specific primers are capable of equally priming thetarget template (cDNA, PCR product, etc.) at high stringency conditions.

The present invention is directed to a process for determining thesequences of the alleles of highly polymorphic gene systems carried byany given individual, such as, for example, the human HLA system, mostparticularly Class I genes, using enzymatic amplification and directsequencing of the gene cDNA molecules using a limited number of primersand avoiding the use of allele specific oligonucleotides as much aspossible. The present method is particularly well suited to determiningallelic sequences of Class I HLA genes, thereby providing complete HLAClass I genotype information for a subject. Using the method of thepresent invention complete Class I HLA typing (HLA-A, -B, and -C) can beperformed in about 16 to 24 hours or less.

Generally, the method of the present invention involves: extraction ofsample nucleic acid; in the case of RNA, generation of cDNA; cDNA orgenomic DNA amplification; direct sequencing of amplification products;and analysis of the direct sequence information. Generation of cDNA,amplifying the cDNA and direct sequencing the cDNA amplificationproducts is accomplished using oligonucleotide primers with specificcharacteristics, such as those described herein.

More specifically, the primers for binding sites on Class I HLAtranscripts and cDNA molecules provided in the present invention allow:a) selection of the alleles at each separate locus (A or B or C) and yetallow the detection of both possible alleles at each locus in unknownheterozygotes; and b) generation of sequencing information encompassingboth polymorphic exons of Class I genes by using the fewest possiblenumber of reactions.

In the case of RNA, cDNA molecules for each allele at each Class I locus(A or B or C loci) on each chromosome that are to be sequenced aresynthesized by employing a locus-specific oligonucleotide primer thatanneals to a region of each gene locus which has a sequence that isshared by all the alleles at a given locus (with up to about twonucleotide mismatches between the primer and any allele at that locus)but is different for all the alleles at the other related loci (with atleast about two mismatches).

The sample nucleic acid sequence is determined by: amplifying the cDNAmolecules by PCR, after removing the unincorporated oligonucleotideprimer used for generating the cDNA molecules (clean), to generatesufficient product for each allele of each gene locus to be sequenced,with all of the alleles for each gene locus and chromosome to besequenced being amplified with at least one Class I loci-specificoligonucleotide primer annealing equally to all possible alleles at eachClass I gene loci at each chromosome and a locus-specificoligonucleotide primer that anneals to a region of each gene locus whichhas a sequence that is shared by all the alleles at the given locus(with up to about two nucleotide mismatches between the primer and anyallele at that locus) but is different for all the alleles at the otherrelated loci (with at least about two mismatches); preparing theproducts of each PCR for sequencing (clean); sequencing directly theproducts of each PCR product to detect each allele at each gene locus ofeach chromosome with Taq polymerase and either a locus-specific primerand a Class I loci-specific oligonucleotide primer or two Class Iloci-specific oligonucleotide primers; and analyzing each sequencedproduct for each locus and primer combination(s) to determine thegenotype of the subject.

A. Oligonucleotide Primers

The oligonucleotide primers of the present invention can be synthesizedusing any known suitable method, such as phosphotriester andphosphodiester methods. Narang et al., Methods Enzymol., 68:90 (1979);Brown et al., Methods Enzymol., 68:109 (1979). Oligonucleotides can beprepared using a modified solid support such as a Biosearch 8750 DNAsynthesizer. Useful primers can also be isolated from a biologicalsource using appropriate restriction endonucleases which cut doublestranded DNA at or near a nucleotide sequence of interest for use as aprimer.

B. Extraction of Sample Nucleic Acid

In the process of the present invention any source of nucleic acid canbe used as the sample nucleic acid, as long as the sample contains thenucleic acid sequence of interest. For example, the sample chosen forthe present method can be RNA, DNA or a DNA/RNA hybrid. While typicalsamples include peripheral blood mononuclear cells, (PBMNC's),lymphoblastoid cell lines (LCL's), hair cells or the like, fordetermining human HLA Class I gene polymorphisms LCL's or PBMNC's arepreferred. The nucleic acid to be isolated (e.g. RNA or DNA) will dependon the source of genetic material (blood stain, hair, or peripheralblood cells). However, in the case of HLA Class I genes including HLA-A,HLA-B and HLA-C, the preferred isolated nucleic acid is total cellularRNA when the typing is to be done for transplantation purposes orpaternity testing. For forensic uses, genomic DNA may be the preferredgenetic material in which case different primer considerations would beused. Cytoplasmic and poly(A) +RNA can also be used. It is envisionedthat isolation of sample nucleic acid for the present process can beautomated using a DNA/RNA extractor (such as Model 341 DNA extractoravailable from Applied Biosystems, Inc.; Foster City, Calif.).

C. Generation of cDNA

Complementary DNA (cDNA) of the sample nucleic acid is generated usingspecific oligonucleotide primers and cloned reverse transcriptasefollowing general conditions suggested by the enzyme manufacturer(Bethesda Research Laboratories, Gaithersburg, Md.). Specificdifferences in type and amount of primers used, dNTP concentrations andelongation times will be readily apparent to those of skill in the artbased on the Examples that follow.

D. Polymerase Chain Reaction

Amplification of cDNA or genomic DNA for each gene locus of interest isaccomplished using the polymerase chain reaction (PCR) as generallydescribed in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis. The PCRconsists of many repetitions of a cycle which consists of: (a) adenaturation step, which melts both strands of a DNA molecule; (b) anannealing step, which is aimed at allowing the primers to annealspecifically to the melted strands of the DNA molecule; and (c) anextension step, which incorporates to the primers deoxyribonucleotidescomplementary to those of the strand of DNA to which the primers areannealed. The PCR process, as indicated in the Examples, can beconducted using a Thermocycler (Perkin-Elmer, Cetus, Emeryville,Calif.).

The conditions used for the PCR reactions will depend on the specificprimers used for a given gene locus application. The concentrations ofprimers, and buffers used will be apparent from and include the processparameters described in the Examples that follow.

E. Direct Sequencing Of PCR Products

Direct sequencing of double-stranded DNA generated by the PCR isaccomplished using Taq polymerase and specific combinations of reagentsat appropriate concentrations. The sequencing procedure can be conductedin an automatic sequencing apparatus such as the 373A Model DNASequencer from Applied Biosystems Inc. (Foster City, Calif.). Thereagents, including sequencing primers, labelled with radioactive ornon-radioactive labels, sequencing conditions, and nucleotidetermination mixtures will be understood by those of skill in the artbased on the direct sequencing procedure specified in the followingExamples. The present sequencing protocol for double-stranded PCRtemplates described here requires the use of specific primers in boththe amplification and sequencing steps. The use of specific primer paircombinations is required to generate clean, recognizable sequenceladders corresponding to each locus to be sequenced that can beinterpreted.

F. Analysis Of Direct Sequenced PCR Products

The nucleic acid ladders resulting from direct sequencing the cDNA orgenomic DNA for each gene locus of interest can be assessed visuallyfrom autoradiograms or by employing a computer programmed withnucleotides sequence information for all alleles of all haplotypes andprocedures for comparing sequenced alleles and known alleles of geneloci of interest. In a preferred embodiment of the present invention,the evaluation of gene locus alleles involves comparison of the genesequences of each polymerase chain reaction product with a library ofknown genotype information such as the information obtained onhomologous cell lines very well characterized by methods other thansequencing [Marsh and Bodmer, Immunogenetics, 31:131 (1990)] as well assequences of individual alleles. This comparison can be conductedvisually or by computer that generates and compares the specificsequence information for each allele of a gene locus.

It is envisioned that the process of the present invention can be usedto amplify and sequence known and unknown highly polymorphic systems(e.g., HLA typing). The method is particularly well suited for Class IHLA typing, reducing its costs, increasing its speed and especiallyimproving its accuracy. The present process is believed to be useful forpaternity testing and forensic medicine, with more accuracy thanrestriction fragment length polymorphism (RFLP), DNA fingerprinting ordot blot-detection systems. While in the latter only a hybridizationpattern is observed, direct sequencing of amplified products shows theexact nucleotide sequence of the amplified genes, and hence is moreaccurate and reliable.

As evidenced by the following Examples, sequence polymorphism analysisof Class I HLA genes can be rapidly performed in any subject of unknownHLA type by means of enzymatic amplification and direct sequencing ofClass I genes using a limited number of locus-specific and Class Iloci-specific oligonucleotide primer combinations. The approachdescribed herein is entirely automatable using currently availabletechnology and, as opposed to previously described methods usingoligonucleotide probes and dot blots, has the advantage of detecting thepresence of new allelic sequences or sequence microheterogeneity at thepopulation level. The methodology of the present invention is envisionedto be useful for detailed analyses of the effects of sequence allelismat different Class I HLA loci on graft survival after allogeneictransplantation. The method of the present invention allows rapid andprecise sequence analysis of Class I HLA polymorphism in studies ofhuman disease and may be of interest in the search for new Class Isequence variants in large populations of subjects.

The present invention is further described by illustration in thefollowing Examples which are not intended to limit the invention.

EXAMPLE I

1. Preparation of Oligodeoxyribonucleotide Primers and Sequence PrimerCombinations Useful for cDNA/PCR/Sequencing Reactions of Class II HLAGenes

All of the oligodeoxyribonucleotide primers described herewithin weresynthesized as described below:

Automated Synthesis of oligodeoxyribonucleotide primers: Theb-cyanoethylphosphoamidites, obtained from Milligen-Biosearch (Novato,Calif.), were sequentially condensed to a nucleoside derivatizedcontrolled pore glass support using a Biosearch 8750 DNA synthesizer.Condensation cycles included detritylation with dichloroacetic acid indichloromethane, followed by condensation with benzotriazole and cappingwith acetic anhydride and 1-methylimidazole in tetrahydrofuran andpyridine, with each cycle time being approximately 9 minutes. Yields ateach step were >99% as determined by measuring dimethoxytrityle alcoholrelease. The methodology for oligodeoxyribonucleotide synthesis isdescribed in Caruthers, et al., Methods Enzymol., 154:287 (1987).

Deprotection and purification of oligodeoxyribonucleotide primers:Deprotection and purification of oligodeoxyribonucleotide primers wasperformed using the procedure described by Schulhof et al., Nucl. AcidsRes., 15:397 (1987). Briefly, the oligodeoxyribonucleotide was removedfrom the solid support by exposure to concentrated ammonium hydroxide atroom temperature for about one hour. The solution containing thepartially deprotected oligodeoxyribonucleotide was brought to 65° C. for16 hours. Ammonia was removed and the residue was subjected tochromatography on a C18 reverse-phase column (RP 304, BioRad, Richmond,Va.) using a linear gradient of 14 to 20% acetonitrile in 0.1 molarammonium/triethylamine, pH 7.0. The dimethoxytrityle group was removedfrom the HPLC-purified oligodeoxyribonucleotide by treatment with 70%acetic acid. The detritylated oligodeoxyribonucleotide was recovered byprecipitation in ether, vacuum centrifuged until dry, resuspended inwater and quantitated by measuring its absorbance at 260 nm.

Using the above procedure, the following oligonucleotide primerscorresponding to specified regions of the Class I HLA-A, HLA-B and HLA-Cloci were synthesized (see Table I below) and extensively tested:

                                      TABLE I                                     __________________________________________________________________________    Oligonucleotides Used for the                                                 cDNA/PCR/Sequencing Reactions of Class I HLA Genes                            Sequence                                                                      Listing                                                                       No. (Seq)                                                                           Primer                    Anneal                                                                             Locus (i)                                                                          Template                                                                           Step                           __________________________________________________________________________    1     ABC 101                                                                            5'-GGCCCTGACCGAGACCTGGGC-3'                                                                        -8/-1                                                                              A/B/C                                                                              RNA  PCR                            2     ABC 102                                                                            5'-AGATGGCTCCCATCTCAGGGT-3'                                                                        271-277                                                                            A/B/C                                                                              RNA  RT                             3     C103 5'-AGGGGCTCTGGCAGCCCCTCG-3'                                                                        270-263                                                                            C    RNA  RT                             4     B104 5'-AGGGGCTTCGGCAGCCCCTCA-3'                                                                        270-263                                                                            B    RNA  RT                             5     A105 5'-AGGGGCTTGGGCAGACCCTCA-3'                                                                        270-263                                                                            A    RNA  RT/PCR                         6     C106 5'-GTCACGTGTGTCTTTGGGTGT-3'                                                                        183-190                                                                            C    RNA  PCR                            7     B107 5'-GGTCACATGTGTCTTTGGGGG-3'                                                                        184-190                                                                            B    RNA  PCR                            8     A108 5'-AGAGATAGCGTGGTGGGTCAT-3'                                                                        189-195                                                                            A    RNA  PCR/SEQ                        9     ABC109                                                                             5'-CAGTGGGCTACGTGGACGACA-3'                                                                         24-31                                                                             A/B/C                                                                              RNA  PCR/SEQ                        10    ABC116                                                                             5'-GTCGCTGTCGAACCTCACGAA-3'                                                                         33-39                                                                             A/B/C                                                                              RNA  SEQ(alt)                       11    ABC112                                                                             5'-TTCAGGGCGATGTAATCCTT-3'                                                                         121-127                                                                            A/B/C                                                                              RNA  SEQ                            12    ABC113                                                                             5'-GGCAAGGATTACATCGCCCTG-3'                                                                        120-126                                                                            A/B/C                                                                              RNA  SEQ                            __________________________________________________________________________

2. Combinations of Primers for cDNA/PCR/Sequence Reactions

There are specific combinations of oligonucleotide primers for eachreaction and for each locus, including cDNA synthesis, PCR amplificationand direct sequencing, which are designed to provide all the necessarysequence information for obtaining highly accurate, fast and inexpensivetyping results. These combinations are listed in Table II below. Each ofthese combinations of oligonucleotides is characterized by its abilityto generate an end-product (sequencing ladder) which is suitable ofbeing accurately read by the naked eye or processed by computer operatedunder appropriate software.

For typing purposes in the clinical setting, such as in transplantation,the method uses RNA isolated from peripheral blood mononuclear cells asstarting material; for forensic purposes, however, DNA is often the onlyavailable template on which amplification and sequencing primers can beemployed. The specific combinations of primers for RNA analysis aredescribed below in more detail. The general overview of the HLA typingstrategy employing these primer combinations is shown in FIGS. 1(A-D)and 2 and discussed further in Example 2.

                  TABLE II                                                        ______________________________________                                        Combinations of Primers for cDNA/PCR/Seq Reactions                            Lo-                                                                           cus      cDNA*    PCR1     PCR2  A.T.**                                                                              Seq                                    ______________________________________                                        (@)1. A      ABC102   ABC109 A105  55° C.                                                                       A108                                 2.    A      A105     ABC101 A108  55° C.                                                                       ABC112***                            3.    B      B104     ABC109 B107  55° C.                                                                       ABC113                               4.    B      B104     ABC101 B107  55° C.                                                                       ABC112***                            5.    C      C103     ABC109 C106  55° C.                                                                       ABC113                               6.    C      C103     ABC101 C106  55° C.                                                                       ABC112***                            ______________________________________                                         (@) Alternative combination for this reaction: A105 (cDNA), ABC109 (PCR1)     A108 (PCR2), ABC113 (Sequencing).                                             *The RT primer is removed after cDNA synthesis by spindialysis.               **The optimal MgCl.sub.2 concentration is different for different primer      combinations (see below). The concentration ratios between the primers is     also different in different reactions (see below).                            ***Primer ABC116 may be used to sequence these products in order to read      polymorphic sequences close to the 5' end of the cDNAs.                  

EXAMPLE II Protocol: HLA Class I "Typing" by Direct Sequencing of HLA-A,HLA-B and HLA-C Genes

1. Cell Lines and Subjects

Lymphoblastoid cell lines (LCLs) of the 10th InternationalHistocompatibility Workshop [Yang et al., Immunobiology of HLA, Vol I:Histocompatibility Testing 87, (1989); Dupont, Hum Immunol., 26, 3(1989)] were provided by Dr. Miriam Segall (University of Minnesota).Cell lines were also established on 4 subjects, 3 of them belonging tothe same family. All these cell lines had been previously serologicallytyped for HLA Class I and Class II antigens and were used to test themethodology presented here. The serological types of each of thesubjects under study were not known to the investigator performing thesequence analysis at the time the analysis was performed. The cell linesand heterozygote subjects tested included: SA (WS#9001), MZ070782(9002), JBUSH (9035), JVM (9039), BH (9046), SAVC (9034), DEM (9007),WJR076 (9012), RML (9016), RSH (9021), H0301 (9055), SPOO10 (9036), TF(family 6025-003), KR (family 6044-008), PC (family 6044-005) and AR(6044-006).

2. HLA Class I (A, B and C) Transcript Amplification Using ClassI-Specific and Locus-Specific (HLA-A or HLA-B or HLA-C Oligonucleotides

Total cellular RNA was prepared from the cell lines by cesium chloridecentrifugation [Chirgwin et al., Biochemistry, 18, 5249 (1979)]. One to5 μg of total cellular RNA was reverse transcribed with Moloney leukemiavirus reverse transcriptase (MLVRT) 200μ (Bethesda ResearchLaboratories) in 50 mM Tris HCl, pH 8.3, 75 mM KCl, 10 mM DTT, 3 mMMgCl₂, in the presence of the ribonuclease inhibitor RNAs in (10 unitsPromega) 75 mM each dNTP in the presence of a limiting amount (5-20 ng)of a locus-specific (A, B and C loci) or a Class I-specificoligonucleotide (A locus) in a 20 μl final volume for 30-45 min. at 37°C. (See Table I for the sequence and specificity of each of the primersand see Table II for the combinations of primers useful for eachreaction). FIGS. 1A-1D show a schematic of the cDNA, PCR, and sequencingproducts generated in each reaction. Note that each pair of reactionsproposed per locus generates sequence information from differentpolymorphic portions of the genes to be sequenced. After the incubationperiod, the volume of the cDNA synthesis reaction was brought up to 400μl and spun-dialyzed using Ultrafree-100 columns (Millipore) in order toremove the unincorporated oligonucleotide molecules. The following wereadded to the retentate (approximately 50 μl): 10 μl of 10X PCR buffer(50 mM KCl, 100 mM Tris-Cl, pH 8.3, 7.5-15 mM MgCl₂, 0.1% gelatin), 8 μlof a mixture containing 1.25 mM of each of the four dNTPs, from 3-6 μlof a 25 mM MgCl2 solution (the final MgCl2 concentration regulates thestringency of the reaction), 75-200 ng of each of two additionaloligonucleotides for each reaction (reactions 2, 3, 4, 5, and 6) or75-200 ng of one primer and 0.75-2 ng of the other primer (reaction 1)and 2 units of Taq polymerase; the final volume was adjusted to 100 μlwith distilled water.

The reaction mixture was subjected to 35 cycles of 1 min at 94° C., 1min at 55° C. and 1 min at 72° C. using a Perkin-Elmer CetusThermocycler [see Saiki et al., supra (1985); Mullis and Faloona, Supra(1987); Saiki eta! ., supra (1986); Scharf et al., supra (1986)]. Theprimers used, their corresponding sequences and the regions to whichthey anneal are shown in Table II. The reactions for each locus areperformed in separate microfuge tubes (FIG. 2).

Referring to FIG. 2, the specific reaction conditions required in eachmicrofuge tube are as follows:

HLA-A Typing

Reaction 1 (sequencing of 3' end of the alpha2 domain encoding exon ofHLA-A).

a) 10 ng of primer ABC102, 3 microg. of RNA.

b) spin dialysis.

c) PCR with 3 microl. of 25 mM MgCl₂, 100 ng of primer ABC109, 1 ng ofprimer A105. 45 cycles (1' at 92° C., 1' at 55° C., 1' at 72° C.).

d) spin dialysis.

e) sequencing with primer A108.

Reaction 2 (sequencing of 5' end of alpha2 domain encoding exon and thealphal domain encoding exon of HLA-A).

10 ng of primer A105, 3 microg. of RNA.

b) spin dialysis.

c) PCR with 5 microl. of 25 mM MgCl₂, 100 ng of primer ABC101, 100 ng ofprimer A108.

d) spin dialysis.

e) sequencing with primer ABC112.

HLA-B Typing

Reaction 3 (sequencing of 3' end of the alpha2 domain encoding exon ofHLA-B).

a) 10 ng of primer B104, 3 microg. of RNA.

b) spin dialysis.

c) PCR with 5 microl. of 25 mM MgCl₂, 100 ng of primer ABC109, 100 ng ofprimer B107.

d) spin dialysis.

e) sequencing with primer ABC113.

Reaction 4 (sequencing of 5' end of alpha2 domain encoding exon and thealphal domain encoding exon of HLA-B).

a) 10 ng of primer B104, 3 microg. of RNA.

b) spin dialysis.

c) PCR with 5 microl. of 25 mM MgCl₂, 100 ng of primer ABC101, 100 ng ofprimer B107.

d) spin dialysis.

e) sequencing with primer ABC112.

HLA-C Typing

Reaction 5 (sequencing of 3' end of the alpha2 domain encoding exon ofHLA-C).

a) 10 ng of primer C103, 3 microg. of RNA.

b) spin dialysis.

c) PCR with 5 microl. of 25 mM MgCl₂, 100 ng of primer ABC109, 100 ng ofprimer C106.

d) spin dialysis.

e) sequencing with primer ABC113.

Reaction 6 (sequencing of 5' end of alpha2 domain encoding exon and thealphal domain encoding exon of HLA-C).

a) 10 ng of primer C103, 3 microg. of RNA.

b) spin dialysis.

c) PCR with 5 microl. of 25 mM MgCl₂, 100 ng of primer ABC101, 100 ng ofprimer C106.

d) spin dialysis.

e) sequencing with primer ABC112.

The reason for the different ratio of primer concentrations for reaction#1 is that the sequencing primer used to sequence that portion of theHLA-A genes works more efficiently when the limiting primer (0.75-2 ng)is consumed in the reaction. Alternatively, the combination of primersfor reaction 1 can be substituted for primers A105 (cDNA), ABC 109(PCR1), A108 (PCR2) and ABC113 (sequencing) and in this case, 75-200 ngof each PCR primer are used.

2. Direct Sequencing of Amplified Products with Tag Polymerase

The reaction mixture (100 μl) was freed of unincorporated dNTPs andexcess of oligonucleotides by spin-dialysis using Ultrafree-100(Millipore) microconcentrators. Approximately one half of the retentate(20 μl) was dried down and resuspended in 15 μl of 1X Taq sequencingbuffer (50 mM Tris-HCl, pH 9, 10 mM MgCl2). Internal oligonucleotideswere used for priming the sequencing of HLA-A, HLA-B, and HLA-C genes,respectively (Table II). Primers for sequencing are listed in Table II.Eight to 100 ng of primer were end-labelled with 10 pmol of gamma-P32labeled ATP (5000 Ci/mmol, 10 μCI/μL) and 5 units of T4 polynucleotidekinase (Promega Biotec) in a 10 μl final volume. Ten ng of primer (1 μl)were added to the sequencing mixture without extraction ofunincorporated labelled ATP, boiled for 5 min., and then left at roomtemperature for ]5 min. Eight units of recombinant Taq polymerase (USB)were added to the mixture. Four μl of the annealed primer/templatemixture were later added to 4 μl of each of the stop nucleotide mixes:a) Term mix ddG: 15 microM each dGTP, dATP, dCTP, dTTP; 45 microM ddGTP;b) Term mix ddA: 15 microM each dGTP; dATP; dCTP, dTTP; 600 microMddATP; c ) Term mix ddT: 15 microM each dGTP, dATP, dCTP, dTTP; 1200microM ddCTP; d) Term mix ddC: 15 microM each dGTP, dATP, dCTP, dTTP;450 microM ddCTP. The reactions were allowed to proceed for twoconsecutive periods of 10 min. at 72°-74° C. After the second cycle,each reaction was chased with 2 μl of a 7.5 μM mixture of ATP, GTP, TTP,CTP, and allowed to proceed for 5 min. After spinning down, the reactionwas stopped by adding 4 ml of 95% (vol/vol) formamide/20 mM EDTA, heatedto 80° C. for 5 min. and loaded on a 0.4 mm thick 6% polyacrylamide/7Murea gel. Electrophoresis was performed at 2500 V for 2 hr, the gelfixed in 5% (vol/vol) glacial acetic acid/5% (v/v) methanol for 15 min,dried, and exposed to Kodak X-Omat film for 4 to 12 hours.

RESULTS

Optimization of the Sequence-Based Typing of HLA Class I PolymorphicGenes in Heterozygous Cells

The designed primers were tested in different combinations in order togenerate the desired information (sequence ladders). The optimalcombinations of primers and their sequences are shown in Table II. Theproposed reactions (1 through 6) are based on their optimization in twoheterozygote subjects belonging to the same family that share one of thetwo chromosomes transmitted from their parents (AR and PC, see Table IIand FIG. 3). It was known to the investigator interpreting thesequencing results that these subjects were heterozygotes, but thespecific serologic types carried by these individuals were not known tothe investigator.

Different primer concentrations, primer combinations including cDNA, PCRand sequencing primers were tested with an initial objective ofobtaining locus-specific ladders. The combinations of primers chosen asoptimal allowed to obtain locus-specific sequence ladders spanning bothof the polymorphic exons of Class I genes. The assessment of thelocus-specificity of these reactions was based on the presence of basesin the ladders generated that are found in all known alleles at a givenlocus, but are absent in all known alleles at the other loci. Examplesof such bases are indicated in FIGS. 4, 5 and 6.

A second objective was that such a strategy should allow simultaneoussequencing of both possible alleles at a given locus. For that purposeand knowing that the tested cells were heterozygous at the A and B loci,the conditions of the reactions were optimized accordingly; primerconcentrations as well as MgCl₂ concentrations in the PCR reaction weremodified according. An example of such an experiment is shown in FIG. 4.Note that at 3 or less μl of MgCl₂ per PCR reaction, one ladder isselected over the second. By increasing the concentration of MgCl₂ perreaction (4-6 μl of 25 mM MgCl₂), both alleles are equally amplifiedwithout disrupting the locus-specificity of the reaction. Therefore, theoptimum conditions are those which provide for clean, reproduciblesequencing ladders corresponding to both polymorphic exons and whichgenerate ladders corresponding to at least one and all of the possiblealleles carried by any subject at the locus under study (HLA-A or HLA-Bor HLA-C). Interpretation of the sequence ladders generated for the Aand B loci from both subjects allowed to predict their respectiveserological reactivities. However, several base-pair substitutions werenoted in the alleles carried by these subjects if compared to previouslydescribed sequences corresponding to alleles with the same serologicalreactivities (FIGS. 4-6). Furthermore, family member 6044-008 (samefamily) who could not be typed completely by serology was sequenced at Aand B loci using the strategy described here. Partial sequence analysisof this individual's A and B genes showed that she was heterozygous atthe A locus (A*2401/A*0101) but homozygous at the B locus (B*0801)(Table III).

Testing of the "HLA Class I Sequence-Based Typing" Strategy inSerologically-Typed Cell Lines

A series of cell lines which had been serologically typed was testedwith the optimized strategy, although this serological information wasnot known to the investigator reading the sequence ladders. The sequenceladder corresponding to each of the performed reactions was first read,compared to known sequences and assigned a serological designationcorresponding to the specificity with the highest sequence homology. Theresults of these experiments are shown in Table III. As shown in TableIII, the serological reactivity of the cells could be predicted in allbut one case; sequencing of the 3'-end of the third exon of the B genes(encoding the alpha2 domain of the heavy chain of the B antigen) carriedby cell line WJR076 indicated that it carried a B*5800 allele. However,serological analysis indicated that it carried a highly related allele:B57 which, in the region sequenced by the corresponding reaction,differs from B58 by a single nucleotide substitution (other differencescan be found upstream). This discrepancy could be explained if thesequenced B gene corresponds to a B57 allele that shares its 3' sequencewith the B58 allele.

The consanguineous homozygous typing cell RSH (which is supposed to behomozygous at all HLA loci) reacted with A68 and A30 antisera, althoughthe sequence corresponding to the 3' end of the third exon of the Agenes corresponded to an A*680x gene. It is possible that upstreamportions of the A gene(s) carried by this cell line share sequences withthe A30 specificity; in this case, the cell line would be homozygous fora sequence variant of A68 with 5' sequences of an A30 specificity.Interestingly, as indicated in Table III, several new sequence subtypesof A and B specificities were identified with these experiments. Thesequencing ladders generated with some of these experiments are shown inFIGS. 4, 5, and 6.

The strategy shown here for Class I HLA typing using SBT (sequence-basedtyping) can be used to predict the serological reactivity of the typedcells and can detect previously unidentified allelic variants.Furthermore, this approach is inexpensive, requires only 6cDNA/PCR/Sequencing reactions and allows interpretation of thenucleotide sequences of both polymorphic exons of Class i genes. Thus,this approach can be used as an independent and highly accurate typingmethod for Class I HLA genes. The Class I HLA typing strategy presentedin this application does not require previous typing information and isthe only current technique that will allow the most detailedcharacterization of Class I polymorphism (at the sequence level) in thepopulation.

                  TABLE III                                                       ______________________________________                                        Cell Line                                                                              Serology    Reaction #(*)                                            (WS#)    A       B       A         B                                          ______________________________________                                        SA       24       7      1 (*2401) --                                         (9001)                                                                        MZ070782 24      14      1 (*2401) 3 (*140x)                                  (9002)                                                                        JBUSH    32      38      1 (*3201) 3 (*380new)                                (9035)                                                                        JVM       2      18      1 (*020x) --                                         (9039)                                                                        BH (9046)                                                                               2      13      1 (*020x) 3 (*1301)                                  SAVC (9034)                                                                             3       7      1 (*0301) 3 (*070new)                                DEM (9007)                                                                              2      57      1 (*020x) 3 (*5701)                                  WJR076    2      57      1 (*020x) 3 (*5801)                                  (9012)                                                                        RML (9016)                                                                              2      51      1 (*020x) 3 (*5101)                                  RSH (9021)                                                                             68/30   42      1 (*680x) 3 (*4201)                                  HO301 (9055)                                                                            3      14      1 (*0301) 3 (*140x)                                  HO301 (9055)                                                                            3      14                4 (*140x)                                  SPOO10    2      44      1 (*020x) 3 (*440x)                                  (9036)                                                                        SPOO10    2      44                4 (*4401)                                  (9036)                                                                        KR        1       8      1 (*0101 +                                                                              3 (*0801)                                                           *2401)                                               TF        1/11    8/7    1 (*0101 +                                                                              3 (*0801 +                                                          *110new)  *0702)                                     (#)PC     1/2     8/51   2 (*010new +                                                                            4 (*080new +                                                        *020x)    *510new)                                   (#)AR     1/24   39/51   2 (*020x +                                                                              4 (*390new +                                                        *240new   *510new)                                   ______________________________________                                         (*) The predicted allele by Sequencebased typing (SBT) are shown in           parenthesis: the sequence ladder corresponding to each of the performed       reactions was first read, compared to known sequences and assigned a          serological designation corresponding to the specificity with the highest     sequence homology. The serological results were not known by the              investigator interpreting the sequence ladders. The new nomenclature of       HLA Class I allelic specificities is used here (for example, the              serological A1 allele is designated as A*0101). In some cases, sequence       variants have been described for a given serological specificity; these       are designated as for example A*1101 and A*1102, etc. In many cases, new      sequence variants have been identified by using the method of the present     invention and have been designated as "new". If the sequence difference       that distinguishes two given variants of a given specificity falls outsid     the region sequenced in a given reaction, the sequencing results are          represented as, for example B*140x (in this particular case the sequence      could correspond to either B*1401 or B*1402).                                 (#) These two subjects are siblings. The new B*51 allelic sequence is         shared by both of them as expected; the B allele carried by the second        chromosome is different in each subject.                                      Underlined alleles indicate discrepancy between serological and sequence      results. In KR, serological results for Class I genes were incomplete; th     second chromosome of this subject could not be typed. However, sequence       analysis indicated that the subject was heterozygous at the A locus.          Consanguineous cell line RSH was found to be homozygous for A*680x.      

EXAMPLE III Class I Typing Strategy for Determining Unknown HLA Type

Routine HLA typing of large populations of individuals for sequencepolymorphisms can be performed by the use of the methodology reportedhere which can also identify previously unknown allelic variants. FIG. 2shows a flow-chart for the protocol used to determine sequence allelismof individuals of unknown HLA types.

1. Employment of Primer Combinations for cDNA, PCR and Direct SequencingUsing RNA as Initial Template

In the present method, a given Class I typing primer is considered to be"locus-specific" or Class I loci-specific not exclusively on the basisof its nucleotide sequence, but also according to its functionalbehavior under specific reaction conditions. More specifically, forsynthesizing cDNA molecules, the present invention provides singlestrand DNA anti-sense oligonucleotide primers that anneal to regionsrelatively conserved (no more than about 2 nucleotide differences) thatfollow a locus-specific nucleotide sequence pattern of the gene mRNAs tobe reverse transcribed, amplified and sequenced. These oligonucleotideprimers include an oligonucleotide sequence that: (1) anneals to aregion (codons 271-277) shared by the alleles at HLA-A, -B, and -C loci(primer ABC102); (2) anneals to a region (codons 263-270) shared by thealleles at the HLA-A locus (primer A105); (3) anneals to a region(codons 263-270) shared by the alleles at the HLA-B locus (primer B104);(4) anneals to a region (codons 263-270) shared by the alleles at theHLA-C locus. These primers are used in 6 different reactions (1 through6 in FIG. 2) at a low concentration (10 ng) and are removed byspin-dialysis after the cDNA synthesis reaction in order to increase thefunctional efficiency of the primers used in the PCR and sequencingreactions. Reactions 1 and 2 are for HLA-A typing, reactions 3 and 4 arefor HLA-B typing, and reactions 5 and 6 are for HLA-C typing.

To amplify cDNA molecules corresponding to each Class I locus, twodifferent oligonucleotide primers are added to each reaction. Thereactions for each locus generate "locus-specific" amplification underthe conditions described in the legend to FIG. 2. Each of thesereactions amplify all the alleles carried by any given individual ateach separate locus. Each combination of primers will generateinformation corresponding to only one locus (HLA-A or HLA-B or HLA-C);in heterozygotes at a given locus the sequencing reactions will generate2 overlapping ladders (each ladder corresponding to each allele at thesame locus on each parental chromosome) and in homozygotes only oneladder will be generated (the ladder corresponds to the very same alleleat the same locus on both parental chromosomes). These primers are: (1)a sense primer (e.g. ABC109), annealing to codons 24 to 31 of thealleles at all loci (reaction 1); (2) an anti-sense primer (e.g. A105),annealing to codons 270-263 of the alleles at the HLA-A locus (reaction1); (3) a sense primer (e.g. ABC101), annealing to codons -1 to -8 ofthe alleles at all loci (reactions 2 through 5); (4) an anti-senseprimer (e.g. A108), annealing to codons 189-195 of the alleles at theHLA-A locus (reaction 2); (5) an anti-sense primer (e.g. B107),annealing to codons 184-190 of the alleles at the HLA-B locus (reactions3 and 4); (6) an anti-sense primer (e.g. C106), annealing to codons183-190 of the alleles at the HLA-C locus (reactions 5 and 6).

Primers useful in direct sequencing the polymerase-chain reactionproducts corresponding to Class I HLA loci include: (1) an anti-senseprimer (e.g. A108), annealing to codons 189 to 195 of the alleles atHLA-A locus. This primer is used to sequence the products of reaction 1and generates sequence information corresponding to the alpha2 encodingexon of HLA-A locus; (2) an anti-sense primer (e.g. ABC112), annealingto codons 120 to 126 of the alleles at all Class I loci. This primer isused to sequence the products of reactions 2 (HLA-A), 4 (HLA-B) and 6(HLA-C) and generates sequence information for the 5' end of thealpha2-encoding exon and all the alphal-encoding exon of Class I genes;(3) a sense primer (e.g. ABC113), annealing to codons i20 to 126 of thealleles at all Class I loci. This primer is used for sequencing theproducts of reactions 3 (HLA-B) and 5 (HLA-C) and generates sequenceinformation for the 3'-end of the alpha2-encoding exon of thecorresponding Class I genes.

2. Procedure for Determining Unknown HLA Type

A subject of unknown HLA type, diseased or not, is to be typed for ClassI HLA polymorphism. From 10 to 50 mL of peripheral blood are drawn. Theperipheral blood mononuclear cells are prepared by centrifugation overFicoll-Hypaque gradients. The cells are then lysed in guanidiumisothyocianate and total cellular RNA prepared using conventionalmethods (either by centrifugation on cesium chloride gradients, whichlasts about 16 hours, or by the guanidiumisothyocianate-phenol-chlorophorm extraction method, which can beperformed in less than 4 hours. See Gouuh, supra (1988); Johns et al.,Anal, Biochem., 180:276 (1989). Otherwise genomic DNA from these cellsor other sources (hair, blood stains, sperm, etc.) can be prepared withconventional methods such as provided by Higuchi, R. in PCR Technology,Erlich, M. (ed.), Stockton Press:31 (1989). HLA-A/B/C, HLA-A, HLA-B, andHLA-C cDNA molecules are synthesized from total RNA using locus-specificprimers. Approximately, one to five micrograms of RNA is reversetranscribed with MoLVRT (reverse transcriptase) and HLA-A, -B, and -Cloci, HLA-A (A105 and/or ABC102), HLA-B (B104) and HLA-C (C103),-specific anti-sense primers in a 20 μl final volume reaction (30-60minute incubation). The reaction for each Class I gene is performed in adifferent tube.

Once these reactions are completed, they are spin dialyzed to removeunincorporated reverse transcriptase primer, and the enzymaticamplification of the respective cDNA molecules is then performed byadding to the retentate of the spin-dialysis, the reagents needed forthe amplification step. This includes the PCR reagents and appropriatecombination of Class I loci-specific and individual locus-specificoligonucleotide primers. This example uses two reactions for HLA-A(tubes 1 and 2), two for HLA-B (tubes 3 and 4), and two for HLA-C (tubes5 and 6). Reactions 1 and 2 incorporate primers ABC109 and A105 orABC101 and A108, respectively. Tubes 3 and 4 incorporate primer B107 andABC109 or B107 and ABC101, respectively, and tubes 5 and 6 incorporateprimer C106 and ABC109 or C106 and ABC101, respectively,

Once completed, the reactions are spun-dialyzed for about 15 minutesusing Ultrafree-100 (Millipore) or similar columns to removeunincorporated primers and dNTPs. The retentate or one half of therecovered retentate for each reaction is then directly sequenced usingTaq polymerase and the primers described in Table II for eachcombination of primers used in the cDNA/PCR reactions using P-32end-labeled (10 minutes) sequencing primers (35 minutes).

The sequencing reactions products are loaded on an acrylamide gel,electrophoresed in 2-3 hours and exposed to X-ray films for 4-12 hours.The gels are read and results from gels are compared to nucleotidesequences corresponding to all possible alleles.

Comparisons can be made visually using the naked eye or using a personalcomputer and a software package including the nucleotide sequences ofall alleles of all haplotypes and routines which indicate how thecomparison is to be performed as well as subroutines which will allowidentification of new allelic sequences.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer ABC101                                   (B) LOCATION: Anneals to codons -8 to -1 of the                               A, B and C transcripts of HLA class I                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GGCCCTGACCGAGACCTGGGC21                                                       Ala LeuThrGluThrTrpAla                                                        5-1                                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                           (v) FRAGMENT TYPE: Internal Fragment                                         (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer ABC102                                   (B) LOCATION: Anneals to codons 271 to 277 of the                             A, B and C transcripts of HLA class I                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      AGATGGCTCCCATCTCAGGGT21                                                       (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer C103                                     (B) LOCATION: Anneals to codons 263 to 270 of the                              C transcript of HLA class I                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      AGGGGCTCTGGCAGCCCCTCG21                                                       (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer B104                                     (B) LOCATION: Anneals to codons 263 to 270 of the                             B transcript of HLA class I                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      AGGGGCTTCGGCAGCCCCTCA2 1                                                      (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer A105                                     ( B) LOCATION: Anneals to codons 263 to 270 of the                            A transcript of HLA class I                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AGGGGCTTGGGCAGACCCTCA21                                                       (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                       (D) TOPOLOGY: Linear                                                         (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer C106                                     (B) LOCATION: Anneals to codons 183 to 190 of the                             C transcript of HLA class I                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GTCACGTGTGTCTTTG GGTGT21                                                      (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                  (A) NAME/KEY: Oligonucleotide Primer B107                                    (B) LOCATION: Anneals to codons 184 to 190 of the                             B transcript of HLA class I                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      GGTCACATGTGTCTTTGGGGG21                                                       (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Single                                                     (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer A108                                     (B) LOCATION: Anneals to codons 189 to 195 of the                             A transcript of HLA class I                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      A GAGATAGCGTGGTGGGTCAT21                                                      (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                           (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                    (ix) FEATURE:                                                                (A) NAME/KEY: Oligonucleotide Primer ABC109                                   (B) LOCATION: Anneals to codons 24 to 31 of the                               A, B and C transcripts of HLA class I                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      CAGTGGGCTACGTGGACGACA21                                                       ValGlyTyrValAspAsp                                                             2530                                                                         (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                    (ix) FEATURE:                                                                (A) NAME/KEY: Oligonucleotide Primer ABC116                                   (B) LOCATION: Anneals to codons 33 to 39 of the                               A, B and C transcripts of HLA class I                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GTCGCTGTCGAACCTCACGAA21                                                       (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                      (B) TYPE: Nucleic Acid                                                       (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: yes                                                          (v) FRAGMENT TYPE: Internal Fragment                                          (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer ABC112                                   (B) LOCATION: Anneals to codons 121 to 127 of the                             A, B and C transcripts of HLA class I                                         (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                    TTCAGGGCGATGTAATCCTT20                                                        (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (iv) ANTI-SENSE: no                                                            (v) FRAGMENT TYPE: Internal Fragment                                         (vi) ORIGINAL SOURCE: Synthetically Derived                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Oligonucleotide Primer ABC113                                   (B) LOCATION: Anneals to codons 120 to 126 of the                             A, B and C transcripts of HLA class I                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GGCAAGGATTACATCGCCCTG21                                                       Gly LysAspTryIleAlaLeu                                                        120125                                                                    

What is claimed:
 1. A method for determining a nucleotide sequence of aClass I HLA-A gene locus of a subject in a sample containing subjectRNA, said method comprising:(a) synthesizing, from RNA isolated fromsaid sample, a cDNA molecule for each allele of said gene locus using ina first synthesis reaction an oligonucleotide primer depicted in SEQ. IDNO: 2 to produce a first cDNA molecule and in a second synthesisreaction an oligonucleotide primer depicted in SEQ. ID. NO:5 to producea second cDNA molecule, wherein in said first and second synthesisreactions said primer is present in a limiting amount; (b) amplifyingsaid first and second cDNA molecules to generate sufficient first andsecond double stranded amplification reaction products respectively forsequencing each allele of said gene locus using in the first amplifyingreaction an oligonucleotide primer depicted in SEQ. ID. NO:9 and anoligonucleotide primer depicted in SEQ. ID. NO:5 and in the secondamplifying reaction an oligonucleotide primer depicted in SEQ. ID. NO:Iand an oligonucleotide primer depicted in SEQ. ID. NO:8; (c) sequencingdirectly each first and second amplification reaction product to producetwo sequencing ladders for each amplification reaction product using inthe first sequencing reaction an oligonucleotide primer depicted in SEQ.ID. NO:8 and in the second sequencing reaction an oligonucleotide primerdepicted in SEQ. ID. NO:11; and (d) analyzing each sequencing ladder todetermine the nucleotide sequence of said HLA-A gene locus.
 2. Themethod according to claim 1, wherein said primer depicted in SEQ. ID.NO:5 is present in said first amplifying reaction in a limiting amountso that it is consumed in said reaction.
 3. A method for determining anucleotide sequence of a Class I HLA-B gene locus of a subject in asample containing subject RNA, said method comprising:(a) synthesizing,from RNA isolated from said sample, a cDNA molecule for each allele ofsaid gene locus using in a first synthesis reaction an oligonucleotideprimer depicted in SEQ. ID NO:4 to produce a first cDNA molecule and ina second synthesis reaction an oligonucleotide primer depicted in SEQ.ID. NO:4 to produce a second cDNA molecule, wherein in said first andsecond synthesis reactions said primer is present in a limiting amount;(b) amplifying said first and second cDNA molecules to generatesufficient first and second double stranded amplification reactionproducts respectively for sequencing each allele of said gene locususing in the first amplifying reaction an oligonucleotide primerdepicted in SEQ. ID. NO:9 and an oligonucleotide primer depicted in SEQ.ID. NO:7 and in the second amplifying reaction an oligonucleotide primerdepicted in SEQ. ID. NO:I and an oligonucleotide primer depicted in SEQ.ID. NO:7; (c) sequencing directly each first and second amplificationreaction product to produce two sequencing ladders for eachamplification reaction product using in the first sequencing reaction anoligonucleotide primer depicted in SEQ. ID. NO:12 and in the secondsequencing reaction an oligonucleotide primer depicted in SEQ. ID.NO:11; and (d) analyzing each sequencing ladder to determine thenucleotide sequence of said HLA-B gene locus.
 4. A method fordetermining a nucleotide sequence of a Class I HLA-C gene locus of asubject in a sample containing subject RNA, said method comprising:(a)synthesizing, from RNA isolated from said sample, a cDNA molecule foreach allele of said gene locus using in a first synthesis reaction anoligonucleotide primer depicted in SEQ. ID NO:3 to produce a first cDNAmolecule and in a second synthesis reaction an oligonucleotide primerdepicted in SEQ. ID. NO:3 to produce a second cDNA molecule, wherein insaid first and second synthesis reactions said primer is present in alimiting amount; (b) amplifying said first and second cDNA molecules togenerate sufficient first and second double stranded amplificationreaction products respectively for sequencing each allele of said genelocus using in the first amplifying reaction an oligonucleotide primerdepicted in SEQ. ID. NO:9 and an oligonucleotide primer depicted in SEQ.ID. NO:6 and in the second amplifying reaction an oligonucleotide primerdepicted in SEQ. ID. NO:i and an oligonucleotide primer depicted in SEQ.ID. NO:6; (c) sequencing directly each first and second amplificationreaction product to produce two sequencing ladders for eachamplification reaction product using in the first sequencing reaction anoligonucleotide primer depicted in SEQ. ID. NO:12 and in the secondsequencing reaction an oligonucleotide primer depicted in SEQ. ID.NO:11; and (d) analyzing each sequencing ladder to determine thenucleotide sequence of said HLA-C gene locus.
 5. A method as in any ofclaims 1-4, for rapid automated determination of HLA Class I genotype ofa subject in a sample containing subject nucleic acid wherein:(a) saidRNA is isolated from said sample with an RNA/DNA extractor; (b) saidamplifying reactions are done using a thermocycler; (c) said sequencingis done using an automated sequencing apparatus; and (d) said analyzingeach sequencing ladder to determine the nucleotide sequence of each genelocus of interest of said subject is done with a computer having a database with allelic sequence information to compare the sequence of eachallele of each gene locus sequenced to known sequences for each genelocus of interest, wherein said gene locus is selected from the groupconsisting of HLA-A, HLA-B, and HLA-C.
 6. An oligonucleotide primerhaving the sequence depicted in SEQ. ID NO:
 2. 7. An oligonucleotideprimer having the sequence depicted in SEQ. ID NO:3.
 8. Anoligonucleotide primer having the sequence depicted in SEQ. ID NO:4. 9.An oligonucleotide primer having the sequence depicted in SEQ. ID NO:5.10. An oligonucleotide primer having the sequence depicted in SEQ. IDNO:6.
 11. An oligonucleotide primer having the sequence depicted in SEQ.ID NO:7.
 12. An oligonucleotide primer having the sequence depicted inSEQ. ID NO:
 8. 13. An oligonucleotide primer having the sequencedepicted in SEQ. ID NO:9.
 14. An oligonucleotide primer having thesequence depicted in SEQ. ID NO:10.